Fall 2013 - Week 10 & 11 Cell Lysis, Protein Purification and Characterization (10/13/13)
The DH5a cells containing the DHFR protein were lysed with a sonicator. The enzyme was purified using a Ni-NTA resin column, with a buffer of 300mM imidazole as the elution solution. A PAGE gel was run to verify proper purification of the protein, it was stained, and left to destain overnight. Large-Scale Expression (10/09/13)The small overnight cultures were grown in a larger volume, activated with IPTG, and centrifuged for protein harvesting. The pellet with a weight of 2.45g, was stored in the -80°C freezer. Growth and Small Overnight Culture (10/08/13)
Many colonies were observed growing on the plate of transformed cells. Two small overnight cultures were created using two colonies from the transformation plate (10/07/13), and allowed to grow overnight at 37ºC. Transformation of DH5a (10/07/13)Pre-isolated plasmids containing the E. coli DHFR gene were transformed into competent DH5a cells. The cells were plated on an LB+Kan plate, and allowed to grow overnight at 37ºC. Surrogate Enzyme (10/05/13)Attention was switched from the DHFR-TS of L. major drug target, to the surrogate enzyme target DHFR of E. coli so as to make positive forward progress in lab.
Fall 2013 - Week 7 & 8
Good work Tony. Nice captions and analysis. Thank you. -Max 10/21/13
Transformed Cell Growth (10/19/13)Growth of the transformed DH5a cells was observed on the morning of 10/19. Plates were stored at -20ºC until a master plate could be created. Cohesive End Generation, Annealing, and Transformation (10/18/13)Cohesive end generation of PCR² (09/06) and cut pNIC-Bsa4 was performed. This was followed by annealing and transformation into Competent DH5a cells. The transformed E.coli was plated onto two Kan+Sucrose plates, and left to incubate overnight at 37º C. Virtual Screening of ChemBridge Diversity Library (10/17/13)A screening job was submitted for docking of the ChemBridge Diversity Library of 50k compounds in the homology mode active site. Cut pNIC-Bsa4 plasmid was cleaned using Sigma PCR Clean-Up Kit. Final concentration ~40.0ng/uL
Figure 39 - Absorption Spectrum of cut and cleaned pNIC-Bsa4 plasmid at 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
Crystal Structure of DHFR-TS (L. major) Received (10/15/13)A crystal structure for DHFR-TS (L. major) was received via email from Dr. David Matthews. This structure will be modified and prepared for all future virtual screening runs. Structure available in student folder (Google Drive).
PCR Squared Performed (10/14/13)PCR Squared was performed, and a 1% agarose gel was run. The sample showed too much contamination in the form of multiple unwanted bands. This sample will not be used.
Figure 38 - 1% Agarose gel of PCR Squared; Lane 1: 1kB Ladder, Lane 2: PCR Squared Product using custom forward and reverse primers
GOLD Validation With Control Library (10/11/2013) A control docking was performed on the 3INV_A homology model in order to validate docking using GOLD.
Sequencing Results (10/10/13)Sequencing results for three colonies were received. None were better than 10% match against the expected nucleotide sequence. Sequencing Request (10/08/13)Overnight cultures of the 3 colonies were spun down, mini-prepped, and sent for sequencing using pLIC-For and pLIC-Rev as primers. Small Overnight Cultures, and Midi-Prep (10/07/13)The 3 growing colonies were used to begin small overnight culture. The pNIC-Bsa4 pellet (10/06) was midi-prepped, for a final concentration of ~58ng/uL.
Figure 37 - Absorption Spectrum of midi-prepped pNIC-Bsa4 plasmid at 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
Colony Growth, and Spin-Down (10/06/13)Three colonies were observed to be growing on the overnight plate. It was left overnight again to provide opportunity for further growth. pNIC-Bsa4 culture was spun down, decanted, and stored at -20ºC until mid-prep was possible. Cloning and New pNIC Colony (10/05/13)Cohesive end generation was performed on both pNIC and the gel-extracted PCR Squared product, following cloning protocol. The transformed cells were plated on a Kan+Sucrose agar overnight. Another starter culture of pNIC-Bsa4 was created in 160mL of LB. Left in the shaking incubator overnight. Fall 2013 - Week 5 & 6 Nice Job maintaining your page for these weeks. Keep up the good work. Try to add more information to your nanodrop captions. Thank you.-Max 10/07/2013 Second Round of Mass PCR Squared & Midi-Prep of pNIC (10/03-10/04/13)On 10/03, four PCR Squared reactions were performed using the 2º PCR product from 09/25. On 10/04, four 1% agarose gels with ten wells each were created and placed into four separate electrophoresis rigs. The PCR Squared product was combined and prepped with loading gel. 27uL of this solution was loaded into each well. After running, the bands at ~1,500kB were excised and Gel Extraction was performed using a GenElute Kit. Despite the careful attention to detail, and troubleshooting based on 10/20/13 attempt, the concentration was ~20ng/uL. On 10/04, the overnight culture pNIC-Bsa4 plasmid was midi-prepped, for a final concentration of ~6 ng/uL. The low concentration was most likely a result of a technical error during the procedure.
Figure 36 - Absorption Spectrum of midi-prepped pNIC-Bsa4 plasmid at 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
Figure 35 - Absorption Spectrum of Gel-Extracted L. major DHFR-TS Gene; 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
Figure 34 - 1% agarose gel electrophoresis; Lane 1-9: PCR Squared Product; Lane 10: NEB 1kB Ladder
Overnight Culture of pNIC-Bsa4 (10/03/13)A colony from the cloned pNIC-Bsa4 was grown in 160mL of LB overnight in the shaking incubator. Cloning of pNIC-Bsa4 Into Competent Cells & Docking NADP Into Homology Model Using ICM (10/02/13)pNIC-Bsa4 plasmid was cloned into competent DH5-a cells and plated on a Kan/Agar petri dish. Colonies were allowed to grow overnight. Protocol for docking a ligand into the active site of a .PDB structure using ICM was followed to dock NADPH into the active site of homology model 3invA. Attempts to run a docking script were unsuccessful. Help from the Research Educator was sought.
Figure 33 - Overnight growth of pNIC-Bsa4-cloned DH5-a cells
Docking Attempt of NADP Into Homology Model Using Hermes/GOLD (10/01/13)Docking of NADPH into the the homology model was attempted on the early morning of 10/01, only to discover that the license for the Hermes program had expired the day before. Help from the Research Educator was sought. Ligand Preparation of Control Library (09/29/13)The control ligands from 09/27 were concatenated into a single .sdf file. ICM was used to ligand-prep the library for docking. Creating a Control Library (09/27/13)A positive and negative control library of 17 ligands was created using protocol "ProtocolVirtualScreenYOURTarget_VDS_Fall13_PART1.doc". The library is still to be prepared for docking. All physico-chemical properties of ligands were recorded and made available on the Target Page for DHFR-TS L. major. A list of the chosen control ligands follows:
Fall 2013 - Week 3 & 4 Tony - great job on Homology and PCR squareds - keep driving fowrard with the cloning - we need to get your clone soon so that you guys can move forward - Dr. B 100113 Mass PCR Squared (09/19-20/13)On 09/20, four PCR Squared reactions were performed using the 2º PCR product from 09/02. On 09/21, four 1% agarose gels with six wells each were created and placed into four separate electrophoresis rigs. The PCR Squared product was combined and prepped with loading gel. 50uL of this solution was loaded into the wells of the agarose gels. After running, the bands at ~1,500kB were excised and Gel Extraction was performed using a GenElute Kit. The elution from this extraction (concentration: 77.9 ng/uL) was cleaned using a Sigma PCR CleanUp kit. The low yield of DNA (3.6 ng/uL) resulted in the discarding of the newly-cleaned elution.
Figure 32 - Absorption Spectrum of PCR Cleaned Gel Extraction; 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
Figure 31 - Absorption Spectrum of Gel-Extracted L. major DHFR-TS Gene; 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
Figure 30 - 1% agarose gel electrophoresis; Lane 1: NEB 1kB Ladder; Lane 2-6: PCR2 product of L. major DHFR-TS gene
Creating a Homology Model (09/18/13)Two custom homology model was created for the L. major DHFR-TS enzyme using the ProMol software. PDB entries 3invA and 3irmB were utilized as the template off of which the models were created. The homology structure synthesized using 3invA was the better of the two models, as indicated by the MolProbity score of 2.61. This model will be the basis for initial virtual screening trials.
Figure 29 - Alignment of custom homology model for L. major DHFR-TS (shown as green), and template enzyme 3invA (shown as cyan); RMS = 0.000
pNIC-Bsa4 Culture (09/13-14/13)A1 culture of pNIC-Bsa4 was grown in 160mL of LB overnight. The following morning it was determined that no proliferation had occurred - most likely due to the age of the plate from which the colony was taken. Swiss Model (09/10/13)A Swiss Model screening was performed on the known L. major DHFR-TS Amino Acid sequence. The program determined that PDB entires 3invA and 3irmB would be suitable for the creation of a homology model. Fall 2013 - Week 1 & 2 Good - Dr. B 090913 PCR Squared (09/06/13)PCR² was performed on the 2º PCR product from 09/02/13. A gel was run to confirm that the band was amplified and at the correct nucleotide length
Figure 28 - PCR Squared of Secondary PCR Product; Lane 1: NEB 1kB ladder; Lane 2, 3, 4, 5: PCR Squared product; Lane 6: Empty
Primer Re-Ordering, Primary PCR, Secondary PCR (09/02/13)An new oligo mix was created from primers D4-G5 from primer box B. Primary PCR was performed using the new oligo mix. Secondary PCR was performed using custom primers ordered on 07/03/13. A band was present at ~1500 nucleotides - corresponding with the target gene length of 1590 nucleotides. Samples were stored at -20ºC until PCR² was ready to be performed.
Figure 27 - Overlap PCR of L. major DHFR-TS; Lane1: 100bp Ladder, Lane 2: 1º PCR, Lane 3: 2º PCR, using custom forward and reverse primers, Lane 5: (2) 1º PCR, Lane 6: (2) 2º PCR, using custom forward and reverse primers
Week8
Sequencing Results from Master Plate #2 (07/24/13)The Colony 9 Reverse sequence confirmed the fact that there were two deletions near the second half of the gene. Master Plate #2 results showed only Colony 18 as a promising plasmid. However, there was one point mutation that was not silent.
Colony 9 Reverse Sequence (07/23/13)A sequencing request was placed for Colony 9 using pLic-Rev as a final confirmation that it was not a positive clone.
Blast of Colony 9 Plasmid & Miniprep #2 (07/22/13)CORE Sequences (Forward and Reverse) for Colony 9 were received. The Reverse Sequence was "reverse complemented" and joined with the forward sequence at the points where overlap occurred. The entire sequence was blasted against the known gene. The Blast returned a 100% Query Coverage and a 99% Identity Match. There were two areas of deletion and one point mutation. The clone was negative.
Master plate tubes from 07/19/13 were Miniprepped. Concentrations ranged from 34.45-89.50 ng/uL. Samples of the 6 colonies with the highest concentrations were sent for sequencing using pLIC-For as the primer (Colonies 21, 19, 18, 15, 17, and 24). Secondary samples of colonies 21, and 19 were sent with pLIC-Rev as the primer.
Week 7 Harvesting and Colony 9 Digest (07/19/13)The culture tubes were spun down, and the supernatant was poured out. The tubes were stored in the -20ºC freezer. A digest of only Colony 9 plasmid was performed using XmnI and a higher concentration of plasmid in order to visualize the smallest band. No valuable information was obtained from the digest gel.
Figure 25 - Digest of colony 9 plasmid using XmnI; Lanes 3, 6: 1kb ladder; Lanes 5, 8: Digested colony 9 plasmid; Lane 4: digested, non-cloned pNIC-Bsa4
Sequencing Results & Master Plate 2 (07/18/13)The resulting CORE sequences were blasted against the known gene sequence. Colony 9 resulted in the highest query coverage and identity match (47% Coverage, 96% Identity). An overnight culture of colony 9 was grown overnight. A second master plate and corresponding starter culture tubes were created.
Figure 24 - Blast of Colony 9 cloned plasmid against known gene sequence; using only data from pLIC-For
Master Plate Digest (07/17/13)A digest of the transformed pNIC-Bsa4 plasmid was performed using AcuI as the restriction enzyme. A plasmid containing at least some portion of the gene would result in 3 bands when digested. A non-cloned plasmid would result in 2 bands. All plasmids only showed two bands, however this may have likely been a result of a low amount of plasmid, not allowing the smallest band to be seen.
Figure 24 - Digest of 8 Master Plate plasmids using XmnI; Lane 1: 1kb ladder; Lanes 2-9: varying Master Plate colony plasmids; Lane 10: non-cloned pNIC-Bsa4
Master Plate Sequencing (07/16/13)Samples of the 6 colonies with the highest concentrations were sent for sequencing using pLIC-For as the primer (Colonies 3, 2, 6, 1, 9, and 10). Secondary samples of colonies 3, and 2 were sent with pLIC-Rev as the primer.
Presentation (07/15/13)Presented during weekly meeting about Journal Club article. No lab time.
Week 6
Harvesting & Miniprep (07/12/13)The Master Plate tubes were spun down and purified using the Miniprep Kit from Sigma. Concentrations ranged from 19.1-46.6 ng/uL.
Master Plate and Colony & Overnight Culture (07/12/13)A pair of master plates was created in the evening. 12 tubes corresponding to the master plate squares were prepared and left in the 37ºC shaking incubator overnight.
Figure 23 - pNIC-Bsa4 cloned with DHFR-TS gene, transformed into DH5a cells; grown in a 37ºC incubator overnight on LB Agar + Kan+ Sucrose plate; 4uL Accepting Vector + 8uL Insert
Figure 22 - pNIC-Bsa4 cloned with DHFR-TS gene, transformed into DH5a cells; grown in a 37ºC incubator overnight on LB Agar + Kan+ Sucrose plate; 2uL Accepting Vector + 4uL Insert
Transformation #2 (07/11/13)No growth occurred overnight, so plates were discarded and transformation was attempted again.
Figure 21 - pNIC-Bsa4 cloned with DHFR-TS gene, transformed into DH5a cells; grown in a 37ºC incubator overnight on LB Agar + Kan+ Sucrose plates; Left plate: 2uL Accepting Vector + 4uL Insert; Right plate: 3uL Accepting Vector + 6uL Insert
Transformation #1 (07/10/13)Cohesive end generation of PCR² and cut pNIC-Bsa4 was performed. This was followed by annealing and transformation into Competent DH5a cells. The transformed bacteria was plated onto two Kan+Sucrose plates, and left to incubate overnight at 37º C.
PCR Cleanup, pNIC Digest (07/09/13)PCR² product was cleaned using Sigma PCR Cleanup Kit. The product had an average concentration of 118.6 ng/uL.
Figure 20 - Absorption Spectrum of clean PCR² of DHFR-TS at 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
pNIC-Bsa4 was digested using BsaI-HF, then purified using a Cleanup Kit. The resulting average concentration was 100.4 ng/uL.
Figure 19 - Absorption Spectrum of RE Digested pNIC-Bsa4 at 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
Custom 2º PCR (07/08/13)2º PCR was performed using custom primers that were ordered on 07/03/13. A band was present at ~1500 nucleotides - corresponding with the target gene length of 1590 nucleotides. PCR² was then performed on the 2º PCR product.
Figure 18 - Overlap PCR of L. major DHFR-TS; Lane1: 100bp Ladder, Lane 2: 2º PCR, using custom forward and reverse primers.
Week 5
Midi-Prep of pNIC-Bsa4 (07/05/13)An overnight culture of pNIC-Bsa4 was prepared on 07/02, spun down and frozen at -20°C on 07/03. Midi-Prep was performed on 07/05, resulting in an average concentration of 38 ng/μL, a 260/280 value of 1.91, and a 260/230 value of 2.85. A sample was sent to sequencing on 07/05 using pLIC-for as the primer.
Figure 17 - Absorption Spectrum of pNIC-Bsa4 plasmid at 230nm wavelength using NanoDrop in nucleic acid mode, Trial 2
Figure 16 - Absorption Spectrum of pNIC-Bsa4 plasmid at 230nm wavelength using NanoDrop in nucleic acid mode, Trial 1
PCR Primer Overlap with Q5 Polymerase (07/02/13-07/03/13)An oligo mix was created from primers D4-G5 from primer box B. Primary PCR was performed using the oligo mix. Secondary PCR was performed using primers D4 and G5 as the 'tail primers'.
Figure 15 - Primer Overlap PCR of L. major DHFR_TS; Lanes 1, 6: 100bp Ladder, Lane 2: 1° PCR, Lane 3: 2° PCR, using primers D4 and G5 as 'tail primers'
Tail Primer Design (07/01/13)Forward and reverse primers were designed for the DNA sequence coding for Dihydrofolate Reductase-Thymidylate Synthase in L. major.
Figure 14 - Restriction Enzyme Digest of pGBR22 plasmid; Lanes 1,6: 1kb Ladder, Lanes 2,7: Uncut pGBR22; Lanes 3,8: pGBR22 cut with EcoEI; Lanes 4,9 pGBR22 cut with PvuII; Lanes 5,10 : pGBR22 cut with EcoRI + PvuII
Protein Sonification, Purification, and Characterization (06/26/13 -06/27/13)
Figure 13 – SDS-PAGE Gel Electrophoresis of His-Tag purified PfDXR in pNic-BSSA4; Well 1: Thermo Scientific Prestained Protein Ladder 10-170kDA; Well 2: Cell lysate before Induction; Well 3: Cell lysate after induction; Well 4: Supernatant of Lysed Cells; Well 5: Flowthrough of supernatant flushed with Ni-NTA resin/buffer mix; Well 6: Wash fraction with 20 mM Imidazole; Well 7: Elution 1, using 250mM Imidazole; Well 8: Elution 2, using 250mM Imidazole
Figure 12 - Absorption Spectrum of PfDXR plasmid in Elution 1 at 230nm wavelength using NanoDrop in nucleic acid mode
Figure 11 - Absorption Spectrum of PfDXR plasmid in Elution 2 at 230nm wavelength using NanoDrop in nucleic acid mode
Making an SDS-Page Gel (06/25/13)Gels were made according to the SDS-Page Gel protocol, with a bottom resolving layer and an upper stacking layer. The first gel that was made had a very wide upper stacking layer with bubbles that created large crevices in the wells. A second gel was made the same day and stored at 4°C for use in the protein characterization lab the next day.
Analyzing DNA Sequence (06/24/13)The BLAST database was used to determine the identity of a plasmid DNA sequence sent to the CMB Core. After determining that the sequence was sufficiently similar to that of pGBR22 it was inserted into the DNA sequence of E. coli in the place where it would be cloned into in wet lab. The newly transformed sequence was run through a virtual enzyme digest. The results were compiled to create a virtual gel detailing how various enzymes would cut the new plasmid (i.e. the number and size of bands created).
Week 3
Firefighter Training! (06/19/13)
Figure X - A strapping young lad putting out a very real and very dangerous fire, saving many innocent bystanders
PCR Cloning of pmCHERRYin the MCS Site (06/19/13 - 06/20/13)
Figure 10 – Agarose gel run of pmCHERRY; Lanes 1, 6: 100bp ladder; Lanes 2-5: Dilutions 1-4; Lanes 7-10: Dilutions 5-8
Large-Scale Protein Expression (06/17/13 - 06/18/13)The FabI plasmid in pNIC-Bsa4 was grown in an overnight culture on 06/17, but was not successful. This was likely due to the old age of the colonies used to start the culture. PfDXR in pNic-BSSA4 from another group was used for the Protein Expression protocol. Cells were grown further, activated with IPTG and centrifuged for harvesting. The pellet with a weight of 2.45g, was suspended in buffer and stored in the -80°C freezer.
My Second (and More Successful) PCR! Run of pGBR22 (06/18/13)
Figure 9 – Agarose Gel Run of pgBR22; Lanes 1, 6: 100bp Ladder, Lanes 2, 7: Sample A; Lanes 3, 8: Sample B; Lanes 4, 9: Sample C; Lanes 5, 10: Sample D
Week 2 Mid-Prep of pmCHERRY (06/11/13)
Figure 8 - Absorption Spectrum of pmCHERRY plasmid after midi-prep at 230nm wavelength using NanoDrop in nucleic acid mode; Trial
Figure 7 - Absorption Spectrum of pmCHERRY plasmid after midi-prep at 230nm wavelength using NanoDrop in nucleic acid mode; Trial 2
The final solution yielded an average concentration of 97.8 ng/uL My First PCR! Run of pGBR22 (06/12/13)
Figure 6 - Agarose gel run; Lanes 1 & 6: ladder, Lanes 2-5: pGBR22 (Samples A, B, C, D respectively), Lanes 7-10: pNIC (Samples A, B, C, D respectively)
Week 1
Tony - put in the Weeks in reverse chronological order.Also where is your 1st PCR? of pGBR22.Those plates look pretty dried out - were they in the incubator for 2 days? - DR. B Quantifying DNA Using NanoDrop (06/03/13)
Figure 5 - Absorption Spectrum of pmCHERRY - 120λ at 230nm wavelength using NanoDrop in nucleic acid mode; Trial 1
Figure 2 - Absorption Spectrum of pmCHERRY - 120λ at 230nm wavelength using NanoDrop in nucleic acid mode; Trial 2
Average concentration of pmCHERRY was 119.05 ng/μL Average absorbance at 230nm was .871 Average 260/280 ratio value was 1.91 Average 260/230 ratio value was 2.73
Transformation Efficiency (06/05/13)Images shown in Figures 3-5 were taken on June 10, 2013 - 5 days after being plated. This delay in image capturing accounts for the poor survivorship of the colonies. Colonies were counted the day after plating.
Figure 4 - 1 ng of pmCherry plasmid plated on an LB/Ampicillin plate; 2583 colonies counted; transformation efficiency of 2583 colonies per ng of plasmid
Figure 2 - 5 ng of pmCherry plasmid plated on an LB/Ampicillin plate; 4,500 colonies counted; transformation efficiency of 900 colonies per ng of plasmid
Figure 1 - 25 ng of pmCherry plasmid plated on an LB/Ampicillin plate; 2583 colonies counted; transformation efficiency of 116 colonies per ng of plasmid
Submitting DNA to DNA Sequencing Facility (06/06/13)**
Fall 2013 - Week 10 & 11
Cell Lysis, Protein Purification and Characterization (10/13/13)
The DH5a cells containing the DHFR protein were lysed with a sonicator. The enzyme was purified using a Ni-NTA resin column, with a buffer of 300mM imidazole as the elution solution. A PAGE gel was run to verify proper purification of the protein, it was stained, and left to destain overnight.
Large-Scale Expression (10/09/13)The small overnight cultures were grown in a larger volume, activated with IPTG, and centrifuged for protein harvesting. The pellet with a weight of 2.45g, was stored in the -80°C freezer.
Growth and Small Overnight Culture (10/08/13)
Many colonies were observed growing on the plate of transformed cells. Two small overnight cultures were created using two colonies from the transformation plate (10/07/13), and allowed to grow overnight at 37ºC.
Transformation of DH5a (10/07/13)Pre-isolated plasmids containing the E. coli DHFR gene were transformed into competent DH5a cells. The cells were plated on an LB+Kan plate, and allowed to grow overnight at 37ºC.
Surrogate Enzyme (10/05/13)Attention was switched from the DHFR-TS of L. major drug target, to the surrogate enzyme target DHFR of E. coli so as to make positive forward progress in lab.
Fall 2013 - Week 7 & 8
- Good work Tony. Nice captions and analysis. Thank you. -Max 10/21/13
Transformed Cell Growth (10/19/13) Growth of the transformed DH5a cells was observed on the morning of 10/19. Plates were stored at -20ºC until a master plate could be created.Cohesive End Generation, Annealing, and Transformation (10/18/13)Cohesive end generation of PCR² (09/06) and cut pNIC-Bsa4 was performed. This was followed by annealing and transformation into Competent DH5a cells. The transformed E.coli was plated onto two Kan+Sucrose plates, and left to incubate overnight at 37º C.
Virtual Screening of ChemBridge Diversity Library (10/17/13)A screening job was submitted for docking of the ChemBridge Diversity Library of 50k compounds in the homology mode active site.
Cut pNIC-Bsa4 plasmid was cleaned using Sigma PCR Clean-Up Kit. Final concentration ~40.0ng/uL
PCR Squared Performed (10/14/13)PCR Squared was performed, and a 1% agarose gel was run. The sample showed too much contamination in the form of multiple unwanted bands. This sample will not be used.
GOLD Validation With Control Library (10/11/2013)
A control docking was performed on the 3INV_A homology model in order to validate docking using GOLD.
Sequencing Request (10/08/13)Overnight cultures of the 3 colonies were spun down, mini-prepped, and sent for sequencing using pLIC-For and pLIC-Rev as primers.
Small Overnight Cultures, and Midi-Prep (10/07/13)The 3 growing colonies were used to begin small overnight culture.
The pNIC-Bsa4 pellet (10/06) was midi-prepped, for a final concentration of ~58ng/uL.
Colony Growth, and Spin-Down (10/06/13)Three colonies were observed to be growing on the overnight plate. It was left overnight again to provide opportunity for further growth.
pNIC-Bsa4 culture was spun down, decanted, and stored at -20ºC until mid-prep was possible.
Cloning and New pNIC Colony (10/05/13)Cohesive end generation was performed on both pNIC and the gel-extracted PCR Squared product, following cloning protocol. The transformed cells were plated on a Kan+Sucrose agar overnight.
Another starter culture of pNIC-Bsa4 was created in 160mL of LB. Left in the shaking incubator overnight.
Fall 2013 - Week 5 & 6
Nice Job maintaining your page for these weeks. Keep up the good work. Try to add more information to your nanodrop captions. Thank you.-Max 10/07/2013
Second Round of Mass PCR Squared & Midi-Prep of pNIC (10/03-10/04/13)On 10/03, four PCR Squared reactions were performed using the 2º PCR product from 09/25. On 10/04, four 1% agarose gels with ten wells each were created and placed into four separate electrophoresis rigs. The PCR Squared product was combined and prepped with loading gel. 27uL of this solution was loaded into each well. After running, the bands at ~1,500kB were excised and Gel Extraction was performed using a GenElute Kit. Despite the careful attention to detail, and troubleshooting based on 10/20/13 attempt, the concentration was ~20ng/uL.
On 10/04, the overnight culture pNIC-Bsa4 plasmid was midi-prepped, for a final concentration of ~6 ng/uL. The low concentration was most likely a result of a technical error during the procedure.
Overnight Culture of pNIC-Bsa4 (10/03/13)A colony from the cloned pNIC-Bsa4 was grown in 160mL of LB overnight in the shaking incubator.
Cloning of pNIC-Bsa4 Into Competent Cells & Docking NADP Into Homology Model Using ICM (10/02/13)pNIC-Bsa4 plasmid was cloned into competent DH5-a cells and plated on a Kan/Agar petri dish. Colonies were allowed to grow overnight.
Protocol for docking a ligand into the active site of a .PDB structure using ICM was followed to dock NADPH into the active site of homology model 3invA. Attempts to run a docking script were unsuccessful. Help from the Research Educator was sought.
Docking Attempt of NADP Into Homology Model Using Hermes/GOLD (10/01/13)Docking of NADPH into the the homology model was attempted on the early morning of 10/01, only to discover that the license for the Hermes program had expired the day before. Help from the Research Educator was sought.
Ligand Preparation of Control Library (09/29/13)The control ligands from 09/27 were concatenated into a single .sdf file. ICM was used to ligand-prep the library for docking.
Creating a Control Library (09/27/13)A positive and negative control library of 17 ligands was created using protocol "ProtocolVirtualScreenYOURTarget_VDS_Fall13_PART1.doc". The library is still to be prepared for docking. All physico-chemical properties of ligands were recorded and made available on the Target Page for DHFR-TS L. major. A list of the chosen control ligands follows:
1. DHF (Natural Substrate)
2. 1CX
3. UMP
4. C50
5. MTX
6. TMQ
7. 2CY
8. DQ1
9. WRA
10. P128
11. P65
Fall 2013 - Week 3 & 4
Tony - great job on Homology and PCR squareds - keep driving fowrard with the cloning - we need to get your clone soon so that you guys can move forward - Dr. B 100113
Mass PCR Squared (09/19-20/13)On 09/20, four PCR Squared reactions were performed using the 2º PCR product from 09/02. On 09/21, four 1% agarose gels with six wells each were created and placed into four separate electrophoresis rigs. The PCR Squared product was combined and prepped with loading gel. 50uL of this solution was loaded into the wells of the agarose gels. After running, the bands at ~1,500kB were excised and Gel Extraction was performed using a GenElute Kit. The elution from this extraction (concentration: 77.9 ng/uL) was cleaned using a Sigma PCR CleanUp kit. The low yield of DNA (3.6 ng/uL) resulted in the discarding of the newly-cleaned elution.
Creating a Homology Model (09/18/13)Two custom homology model was created for the L. major DHFR-TS enzyme using the ProMol software. PDB entries 3invA and 3irmB were utilized as the template off of which the models were created. The homology structure synthesized using 3invA was the better of the two models, as indicated by the MolProbity score of 2.61. This model will be the basis for initial virtual screening trials.
pNIC-Bsa4 Culture (09/13-14/13)A1 culture of pNIC-Bsa4 was grown in 160mL of LB overnight. The following morning it was determined that no proliferation had occurred - most likely due to the age of the plate from which the colony was taken.
Swiss Model (09/10/13)A Swiss Model screening was performed on the known L. major DHFR-TS Amino Acid sequence. The program determined that PDB entires 3invA and 3irmB would be suitable for the creation of a homology model.
Fall 2013 - Week 1 & 2
Good - Dr. B 090913
PCR Squared (09/06/13)PCR² was performed on the 2º PCR product from 09/02/13. A gel was run to confirm that the band was amplified and at the correct nucleotide length
Primer Re-Ordering, Primary PCR, Secondary PCR (09/02/13)An new oligo mix was created from primers D4-G5 from primer box B. Primary PCR was performed using the new oligo mix. Secondary PCR was performed using custom primers ordered on 07/03/13. A band was present at ~1500 nucleotides - corresponding with the target gene length of 1590 nucleotides. Samples were stored at -20ºC until PCR² was ready to be performed.
Week 8
Sequencing Results from Master Plate #2 (07/24/13)The Colony 9 Reverse sequence confirmed the fact that there were two deletions near the second half of the gene. Master Plate #2 results showed only Colony 18 as a promising plasmid. However, there was one point mutation that was not silent.
Colony 9 Reverse Sequence (07/23/13)A sequencing request was placed for Colony 9 using pLic-Rev as a final confirmation that it was not a positive clone.
Blast of Colony 9 Plasmid & Miniprep #2 (07/22/13)CORE Sequences (Forward and Reverse) for Colony 9 were received. The Reverse Sequence was "reverse complemented" and joined with the forward sequence at the points where overlap occurred. The entire sequence was blasted against the known gene. The Blast returned a 100% Query Coverage and a 99% Identity Match. There were two areas of deletion and one point mutation. The clone was negative.
Master plate tubes from 07/19/13 were Miniprepped. Concentrations ranged from 34.45-89.50 ng/uL. Samples of the 6 colonies with the highest concentrations were sent for sequencing using pLIC-For as the primer (Colonies 21, 19, 18, 15, 17, and 24). Secondary samples of colonies 21, and 19 were sent with pLIC-Rev as the primer.
Week 7
Harvesting and Colony 9 Digest (07/19/13)The culture tubes were spun down, and the supernatant was poured out. The tubes were stored in the -20ºC freezer.
A digest of only Colony 9 plasmid was performed using XmnI and a higher concentration of plasmid in order to visualize the smallest band. No valuable information was obtained from the digest gel.
Sequencing Results & Master Plate 2 (07/18/13)The resulting CORE sequences were blasted against the known gene sequence. Colony 9 resulted in the highest query coverage and identity match (47% Coverage, 96% Identity). An overnight culture of colony 9 was grown overnight. A second master plate and corresponding starter culture tubes were created.
Master Plate Digest (07/17/13)A digest of the transformed pNIC-Bsa4 plasmid was performed using AcuI as the restriction enzyme. A plasmid containing at least some portion of the gene would result in 3 bands when digested. A non-cloned plasmid would result in 2 bands. All plasmids only showed two bands, however this may have likely been a result of a low amount of plasmid, not allowing the smallest band to be seen.
Master Plate Sequencing (07/16/13)Samples of the 6 colonies with the highest concentrations were sent for sequencing using pLIC-For as the primer (Colonies 3, 2, 6, 1, 9, and 10). Secondary samples of colonies 3, and 2 were sent with pLIC-Rev as the primer.
Presentation (07/15/13)Presented during weekly meeting about Journal Club article. No lab time.
Week 6
Harvesting & Miniprep (07/12/13)The Master Plate tubes were spun down and purified using the Miniprep Kit from Sigma. Concentrations ranged from 19.1-46.6 ng/uL.
Master Plate and Colony & Overnight Culture (07/12/13)A pair of master plates was created in the evening. 12 tubes corresponding to the master plate squares were prepared and left in the 37ºC shaking incubator overnight.
Transformation #2 (07/11/13)No growth occurred overnight, so plates were discarded and transformation was attempted again.
Transformation #1 (07/10/13)Cohesive end generation of PCR² and cut pNIC-Bsa4 was performed. This was followed by annealing and transformation into Competent DH5a cells. The transformed bacteria was plated onto two Kan+Sucrose plates, and left to incubate overnight at 37º C.
PCR Cleanup, pNIC Digest (07/09/13)PCR² product was cleaned using Sigma PCR Cleanup Kit. The product had an average concentration of 118.6 ng/uL.
pNIC-Bsa4 was digested using BsaI-HF, then purified using a Cleanup Kit. The resulting average concentration was 100.4 ng/uL.
Custom 2º PCR (07/08/13)2º PCR was performed using custom primers that were ordered on 07/03/13. A band was present at ~1500 nucleotides - corresponding with the target gene length of 1590 nucleotides. PCR² was then performed on the 2º PCR product.
Week 5
Midi-Prep of pNIC-Bsa4 (07/05/13)An overnight culture of pNIC-Bsa4 was prepared on 07/02, spun down and frozen at -20°C on 07/03. Midi-Prep was performed on 07/05, resulting in an average concentration of 38 ng/μL, a 260/280 value of 1.91, and a 260/230 value of 2.85. A sample was sent to sequencing on 07/05 using pLIC-for as the primer.
PCR Primer Overlap with Q5 Polymerase (07/02/13-07/03/13)An oligo mix was created from primers D4-G5 from primer box B. Primary PCR was performed using the oligo mix. Secondary PCR was performed using primers D4 and G5 as the 'tail primers'.
Tail Primer Design (07/01/13)Forward and reverse primers were designed for the DNA sequence coding for Dihydrofolate Reductase-Thymidylate Synthase in L. major.
Week 4
Restriction Enzyme Digest (06/28/13)
Protein Sonification, Purification, and Characterization (06/26/13 -06/27/13)
Making an SDS-Page Gel (06/25/13)Gels were made according to the SDS-Page Gel protocol, with a bottom resolving layer and an upper stacking layer. The first gel that was made had a very wide upper stacking layer with bubbles that created large crevices in the wells. A second gel was made the same day and stored at 4°C for use in the protein characterization lab the next day.
Analyzing DNA Sequence (06/24/13)The BLAST database was used to determine the identity of a plasmid DNA sequence sent to the CMB Core. After determining that the sequence was sufficiently similar to that of pGBR22 it was inserted into the DNA sequence of E. coli in the place where it would be cloned into in wet lab. The newly transformed sequence was run through a virtual enzyme digest. The results were compiled to create a virtual gel detailing how various enzymes would cut the new plasmid (i.e. the number and size of bands created).
Week 3
Firefighter Training! (06/19/13)
PCR Cloning of pmCHERRYin the MCS Site (06/19/13 - 06/20/13)
Large-Scale Protein Expression (06/17/13 - 06/18/13)The FabI plasmid in pNIC-Bsa4 was grown in an overnight culture on 06/17, but was not successful. This was likely due to the old age of the colonies used to start the culture. PfDXR in pNic-BSSA4 from another group was used for the Protein Expression protocol. Cells were grown further, activated with IPTG and centrifuged for harvesting. The pellet with a weight of 2.45g, was suspended in buffer and stored in the -80°C freezer.
My Second (and More Successful) PCR! Run of pGBR22 (06/18/13)
Week 2
Mid-Prep of pmCHERRY (06/11/13)
The final solution yielded an average concentration of 97.8 ng/uL
My First PCR! Run of pGBR22 (06/12/13)
Week 1
Tony - put in the Weeks in reverse chronological order.Also where is your 1st PCR? of pGBR22.Those plates look pretty dried out - were they in the incubator for 2 days? - DR. B
Quantifying DNA Using NanoDrop (06/03/13)
Average concentration of pmCHERRY was 119.05 ng/μL
Average absorbance at 230nm was .871
Average 260/280 ratio value was 1.91
Average 260/230 ratio value was 2.73
Transformation Efficiency (06/05/13)Images shown in Figures 3-5 were taken on June 10, 2013 - 5 days after being plated. This delay in image capturing accounts for the poor survivorship of the colonies. Colonies were counted the day after plating.
Submitting DNA to DNA Sequencing Facility (06/06/13)**
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